The correlation between markers of oxidative stress and risk factors of coronary artery disease in Thai patients.

An imbalance between oxidative damage and antioxidative protection in association with the pathophysiology of atherosclerosis has been suggested. The aim of our study was to investigate the relationship between plasma lipids, the antioxidant system and oxidative damage in Thai patients with stable coronary artery disease (CAD). Sixty-one patients (40 males, 21 females), who were angiographically defined as having CAD and were clinically stable, participated in this study. Thirty-two healthy subjects (20 males, 12 females) served as normal controls. The investigation included the measurements of plasma lipid profiles and plasma total antioxidative status (TAS) such as plasma vitamin E erythrocyte glutathione (GSH) and glutathione peroxidase (GPx), as well as malondialdehyde (MDA) and total plasma total protein thiols (P-SH). In patients with CAD, erythrocyte GSH and GPx were significantly lower than those found in controls. However plasma TAS and vitamin E were not significantly different between groups. Patients with CAD also had higher MDA and lower P-SH levels than the controls, which represents the oxidative damage products of lipid and proteins. Multiple regression analysis revealed negative correlations between GSH and cholesterol, GSH and low density lipoprotein (LDL), vitamin E and MDA, as well as P-SH and MDA. This study demonstrated the status of oxidative stress in patients with stable CAD. Since oxidative stress is the imbalance between the total oxidants and antioxidants in the body, any single oxidant/antioxidant parameter may not reflect the overall oxidative stress system. Thus, in patients with CAD, diets with various types of antioxidants may be more beneficial in increasing antioxidant activity than any particular antioxidant supplementation.

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.

Potassium status of Northeast Thai constructors in three different geographic locations.

Sudden and unexpected death of young adults during sleep is a phenomenon among Southeast Asians and particularly young Northeast (NE) Thailand constructors in Singapore. Survivor of sudden unexplained death syndrome (SUDS) without structural heart disease with idopathic ventricular fibrillation (VF) has been documented. Low plasma potassium (K) and depletion of K can occur simply through a reduction of K intake and are associated with increased risk of VF. The K-status of the populations was evaluated in the NE (Group 1, n=30), Bangkok (Group 2, n=48) and Singapore (Group 3, n=46). Groups 2 and 3 were further subdivided into Group 2A (worked in Bangkok < or = 1 year, n=8), Group 2B (worked in Bangkok > 1 year, n=40), Group 3A (consumed self-prepared or ready-to-buy meals, n=25) and Group 3B (regularly consumed foods provided free-of-charge by construction companies, n=21). Thirty-four male healthy university personnels from the NE and Bangkok served as the control--Group 4. Two 24-h urine samples and a fasting blood sample were collected from each subject. Dietary-K from food was determined by duplicated meal analysis. All these samples were then analyzed for their K-content. Group 3A had the lowest K-status: their K-intake, serum-K, and urinary-K level were 29 +/- 5.8 mmol/day (% low K-intake=100), 3.43 +/- 0.34 mmol/L (% hypokalemia=48) and 19.23 +/- 8.2 mmol/day (% hypokaliuria=87.5), respectively. Among the construction workers, average K-intake, serum-K and urinary-K levels were 45.5 +/- 6.1 mmol/day (% low K-intake = 37.5), 3.93 +/- 0.2 mmol/L (% hypokalemia = 2.5) and 39.6 +/- 9.2 mmol/day (% hypokaliuria = 12.5), respectively. The values of Group 2B were similar to Group 4. In addition, when the data from all of the groups were compared, there was a positive correlation between dietary-K (intake) and urinary-K (excretion) (r=0.881, p<0.001). In conclusion, NE Thailand constructors from various locations exhibited low K status with low dietary-K, high incidence of hypokalemia, and low urinary-K. From the present study, this low K status may be an important trigger factor for VF in construction workers and associated with increase risk of SUDS.

Ultrastructure and function of mitochondria in gametocytic stage of Plasmodium falciparum.

Morphological properties of the mitochondrial organelles in the asexual and sexual gametocytic stages of Plasmodium falciparum have been analyzed and found to be markedly different. From in vitro cultures of both stages in human erythrocytes, it has been demonstrated that the asexual stages contained a defined double-membrane organelle having a few tubular-like cristae. The numbers of mitochondria in the gametocytes were found to be approximately 6 organelles per parasite, and they showed a greater density of the cristae than that of the asexual stage parasite. The organelles of the gametocytes were successfully purified by differential centrifugation following Percoll density gradient separation with the results of approximately 7% yields and approximately 5 folds. The gametocytic organelles contained much more activities of mitochondrial electron transporting enzymes (i.e., cytochrome c reductase, cytochrome c oxidase) than the asexual stage organelles. Mitochondrial function as measured by oxygen consumption were found to be different between these two stages organelles. Their rates of oxygen consumption were relatively low, as compared to those of human leukocyte and mouse liver mitochondria. In contrast to the coupled mammalian mitochondria, the gametocytic organelles were in the uncoupling state between oxidation and phosphorylation reactions during their respiration. However, they were sensitive to inhibitors of the electron transport system, e.g., antimycin A, cyanide. Our results suggest that the mitochondria of the gametocytic stages are metabolically active and still underdeveloped, although their inner membranes are extensively folded. The biochemical significance of the unique structure of the mitochondria in these developing stages in host erythrocytes remains to be elucidated.

Purification and characterization of Plasmodium falciparum succinate dehydrogenase.

Succinate dehydrogenase (SDH), a Krebs cycle enzyme and complex II of the mitochondrial electron transport system was purified to near homogeneity from the human malarial parasite Plasmodium falciparum cultivated in vitro by FPLC on Mono Q, Mono S and Superose 6 gel filtration columns. The malarial SDH activity was found to be extremely labile. Based on Superose 6 FPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing-PAGE analyses, it was demonstrated that the malarial enzyme had an apparent native molecular mass of 90 +/- 8 kDa and contained two major subunits with molecular masses of 55 +/- 6 and 35 +/- 4 kDa (n = 8). The enzymatic reaction required both succinate and coenzyme Q (CoQ) for its maximal catalysis with Km values of 3 and 0.2 microM, and k(cat) values of 0.11 and 0.06 min(-1), respectively. Catalytic efficiency of the malarial SDH for both substrates were found to be relatively low (approximately 600-5000 M(-1) s(-1)). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with Ki values of 81, 13 and 12 microM, respectively. The malarial enzyme activity was also inhibited by substrate analog of CoQ, 5-hydroxy-2-methyl-1,4-naphthoquinone, with a 50% inhibitory concentration of 5 microM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 microM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 microM. A known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone. had no inhibitory effect on both the malarial SDH activity and the oxygen uptake of the parasite at a concentration of 50 microM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.

Molecular characterization of mitochondria in asexual and sexual blood stages of Plasmodium falciparum.

Molecular mechanisms that regulate gene expression during development of asexual stage to sexual stage of Plasmodium falciparum in the human erythrocyte are largely unknown. There were apparent variations in ultrastructural characteristics of the mitochondrion between the two developing stages. The asexual stage's mitochondrion had developed less than that of the sexual stage. The respiratory complexes of the mitochondrial electron transport system in the asexual stage were approximately 8-10 times less active than those in the sexual stage. Using quantitative polymerase chain reaction to amplify the cytochrome b gene encoding a subunit of mitochondrial cytochrome c reductase, the amount of the cytochrome b gene of the sexual stage was calculated to be approximately 3 times higher than that obtained from the asexual stage. Moreover, using quantitative reverse-transcription polymerase chain reaction, a relatively high level of approximately 1.3-kb transcript mRNA of the cytochrome b gene was observed in the sexual stage compared to the asexual stage. A known single-copy chromosomal dihydrofolate reductase gene was found to have a similar amount in the two stages. These results suggest that the copy number of the mitochondrial gene, including transcriptional and translational mechanisms, plays a major regulatory role in differential expression during the development of the asexual to sexual stage of P. falciparum in the human cell.

Mitochondrial oxygen consumption in asexual and sexual blood stages of the human malarial parasite, Plasmodium falciparum.

The two developmental stages of human malarial parasite Plasmodium falciparum, asexual and sexual blood stages, were continuously cultivated in vitro. Both asexual and sexual stages of the parasites were assayed for mitochondrial oxygen consumption by using a polarographic assay. The rate of oxygen consumption by both stages was found to be relatively low, and was not much different. Furthermore, the mitochondrial oxygen consumption by both stages was inhibited to various degrees by mammalian mitochondrial inhibitors that targeted each component of complexes I- IV of the respiratory system. The oxygen consumption by both stages was also affected by 5-fluoroorotate, a known inhibitor of enzyme dihydroorotate dehydrogenase of the pyrimidine pathway and by an antimalarial drug atovaquone that acted specifically on mitochondrial complex III of the parasite. Moreover, antimalarials primaquine and artemisinin had inhibitory effects on the oxygen consumption by both stages of the parasites. Our results suggest that P. falciparum in both developmental stages have functional mitochondria that operate a classical electron transport system, containing complexes I-IV, and linked to the pyrimidine biosynthetic pathway.

Mitochondrial ubiquinol-cytochrome c reductase and cytochrome c oxidase: chemotherapeutic targets in malarial parasites.

In order to demonstrate that the mitochondrial electron transport system may be a target for antimalarial drug design in the human malarial parasite Plasmodium falciparum, ubiquinol-cytochrome c reductase and cytochrome c oxidase were purified from mitochondria of the parasite cultivated in vitro. It was found that the catalytic efficiency of the two enzymes from the malarial parasite were markedly lower than those from mouse liver mitochondria. The classical inhibitors affecting different quinone binding sites of the mammalian reductase, antimycin and myxothiazole, which had little antimalarial activities on P.falciparum growth in vitro, were found to exhibit little inhibitory effect against the parasite reductase. The malarial parasite reductase was more sensitive to inhibition by the antimalarial drug, 2-[trans-4-(4'-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone, than the mammalian enzyme, suggesting both the therapeutic potential of the target and the drug.

Chemical synthesis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-known target for pyrimethamine and cycloguanil. The low amounts of enzyme obtainable from parasites or the currently available heterologous expression systems have thus far hindered studies of this enzyme. The 1912-base pair P. falciparum DHFR-TS gene was designed based on E. coli codon preference with unique restriction sites evenly placed throughout the coding sequence. The gene was designed and synthesized as three separated domains: the DHFR domain, the junctional sequence, and the TS domain. Each of these domains contained numerous unique restriction sites to facilitate mutagenesis. The three domains were assembled into a complete DHFR-TS gene which contained 30 unique restriction sites in the coding sequence. The bifunctional DHFR-TS was expressed from the synthetic gene as soluble enzyme in E. coli about 10-fold more efficiently than from the wild-type sequence. The DHFR-TS from the synthetic gene had kinetic properties similar to those of the wild-type enzyme and represents a convenient source of protein for further study. The unique restriction sites in the coding sequence permits easy mutagenesis of the gene which should facilitate further understanding of the molecular basis of antifolate resistance in malaria.

The dihydrofolate reductase domain of Plasmodium falciparum thymidylate synthase-dihydrofolate reductase. Gene synthesis, expression, and anti-folate-resistant mutants.

A 693-base pair gene coding for the 27,132-dalton dihydrofolate reductase (DHFR) domain of the thymidylate synthase-dihydrofolate reductase (TS-DHFR) bifunctional protein of Plasmodium falciparum was designed to have Escherichia coli codon preference and multiple unique restriction sites and was chemically synthesized. The gene was overexpressed (> 50% total cellular protein) in E. coli as insoluble inclusion bodies which could be unfolded and refolded to recover soluble enzyme activity. The refolded DHFR was purified by methotrexate-Sepharose affinity chromatography to give the homogeneous enzyme. Active site titration with methotrexate revealed that the purified protein was fully active. The purified DHFR migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of approximately 30 kDa, and gel filtration showed that the protein is a monomer. The yield of purified enzyme was about 5-6 mg/liter of bacterial culture. Kinetic properties of the purified recombinant DHFR were similar to those reported for wild type bifunctional TS-DHFR. Cassette mutagenesis of the synthetic gene was performed to give the S108N and the N51I + S108N mutants which provided DHFRs analogous to pyrimethamine-resistant mutants found in nature.

Saturation site-directed mutagenesis of thymidylate synthase.

We have subjected 12 different codons of a synthetic Lactobacillus casei thymidylate synthase (TS) gene to saturation site-directed mutagenesis to create amino acid "replacement sets" at each of those positions. The target residues were chosen because they are highly conserved and because they are important for the structure and function of the protein as indicated by solution and structural studies. The mutagenesis procedure involved excision of a fragment of the synthetic gene containing the target codon, followed by its replacement with a mixture of oligonucleotides which code for all 20 amino acids and the amber stop codon. TS mutants were identified by DNA sequencing, and catalytically active mutants were identified by genetic complementation using a Thy- strain of Escherichia coli. Only 3 of the 12 target amino acids examined were essential for TS activity; and of the 125 total mutants identified, 57 were catalytically active. These results point to a high degree of plasticity of TS in accommodating function with structural change.

Mitochondria as the site of action of tetracycline on Plasmodium falciparum.

Rhodamine 123 (Rh 123) was used as a fluorescent probe for the mitochondria of the malarial parasite Plasmodium falciparum. On treatment with tetracycline in vitro, a marked decrease in the percentage of parasites with Rh123 fluorescence in the mitochondria was observed in parallel with an increase in the percentage of parasites with abnormal morphology during onset of decrease in parasitemia. Similar results were obtained, over a shorter time period, with 2,4-dinitrophenol. However, the percentage of parasites with fluorescence did not decrease with increase in parasite abnormal morphology or decrease in parasitemia on treatment with pyrimethamine or cycloheximide. Isoelectric focusing-SDS gel electrophoresis of radiolabelled parasite proteins showed two components of 95 and 85 kDa, the synthesis of which was sensitive to tetracycline, but not cycloheximide. It is concluded that tetracycline exerts its action through the effect on parasite mitochondria and mitochondrial protein synthesis.

Evidence for electrogenic accumulation of mefloquine by malarial parasites.

The uptake of mefloquine and chloroquine by Plasmodium chabaudi-infected mouse erythrocytes was measured in the presence and absence of ionophores and uncoupler in order to distinguish between the pH-dependent and pH-independent absorption of these drugs. Nigericin and CCCP (carbonylcyanide m-chlorophenylhydrazone) were used to relax the proton gradients and electrical potentials across the membranes. It was found that 40-60% of the mefloquine uptake, and 90% of the chloroquine uptake, was pH-dependent, the remainder being due to passive binding to cellular constituents. The distribution ratio of the pH-dependent uptake for mefloquine was about three times greater than for chloroquine. According to the lysosomotropic weak base hypothesis in which the neutral forms of weak bases are assumed to equilibrate across membranes, the mefloquine distribution should be smaller than the chloroquine distribution: since mefloquine is singly charged and chloroquine is doubly charged, the chloroquine distribution ratio should vary as the square of the mefloquine ratio. We interpret the greater uptake ratio of mefloquine to be evidence for the involvement of secondary active transport, with drug uptake being coupled to proton outflow by an antiporter protein. It is proposed that the uptake of mefloquine is electrogenic, with the proton gradient and the electrical potential both contributing to the driving force, but that the proton gradient alone is responsible for the chloroquine uptake.

Depression of Plasmodium falciparum dihydroorotate dehydrogenase activity in in vitro culture by tetracycline.

The activity of Plasmodium falciparum dihydroorotate dehydrogenase, a particulate, electron transport-linked enzyme involved in de novo pyrimidine synthesis, was depressed when the parasite was cultured in the presence of a therapeutic concentration of tetracycline over a 96 h period. There was no direct inhibitory effect of the antibiotic on the enzyme activity. The activity of glutamate dehydrogenase, which is cytoplasmic in the parasite, was unaffected by tetracycline over the same period. Dihydroorotate dehydrogenase activity was substantially recovered when electron acceptors were added. It is suggested that the effect of tetracycline is manifested at the level of the dehydrogenase and/or the electron transport chain linked to this enzyme.